CN-121992065-A - In-vitro activity evaluation method of bactericide for preventing and treating red-shell fungus

CN121992065ACN 121992065 ACN121992065 ACN 121992065ACN-121992065-A

Abstract

The application relates to the technical field of plant protection and discloses an in-vitro activity evaluation method of a bactericide for preventing and treating chaetomium robustum, which comprises the steps of preparing a PDA (personal digital assistant) culture medium and sterilizing, preparing a standardized bacterial cake after activating the chaetomium robustum, adding a medicament into the culture medium according to a preset concentration gradient to prepare a medicament-containing flat plate, inoculating the bacterial cake to the center of the flat plate, culturing under the condition of constant temperature and darkness, measuring the diameter of a bacterial colony and calculating the inhibition rate, establishing a virulence regression equation to obtain an EC50 value, and finally dividing the medicament into high-efficiency, medium-activity and low-efficiency ineffective classes according to the EC50 value. According to the application, by constructing a standardized in-vitro culture system and a quantitative evaluation standard, the interference of environmental and bacterial age differences on experimental results is effectively avoided, the dominant medicament aiming at the red-shell fungus in the strong soil can be rapidly and accurately screened, and reliable data support is provided for the accurate prevention and control of the stem basal rot of the schisandra chinensis.

Inventors

XU PEILEI
YANG YIMING
LI JIAQI
LU WENPENG
Tian taiping
ZHU JINGMENG
WANG YUE
WANG YANLI
HAN YONGLIN
LI CHANGYU
QIN HONGYAN
FAN SHUTIAN

Assignees

中国农业科学院特产研究所

Dates

Publication Date: 20260508
Application Date: 20260226

Claims (10)

1. An in-vitro activity evaluation method of a bactericide for preventing and treating strong red-shelled bacteria is characterized by comprising the following steps of: S1, preparing a culture medium, namely preparing a PDA culture medium for the growth of the chaetomium robustum, sterilizing and cooling for later use; s2, preparing a fungus cake, namely inoculating the strong soil red shell fungus to a PDA culture medium for activation culture, and taking a standardized fungus cake with vigorous hypha growth; s3, preparing a medicine-containing flat plate, namely preparing a to-be-detected bactericide into mother liquor, and respectively adding the mother liquor into the PDA culture medium in the step S1 according to a preset concentration gradient to prepare medicine-containing flat plates with different concentrations; S4, inoculating and culturing, namely inoculating the standardized bacterial cake prepared in the step S2 to the centers of the drug-containing flat plate and the blank control in the step S3, and culturing under the condition of constant temperature and darkness; S5, determining and analyzing results, namely respectively measuring the colony diameters of each concentration gradient drug-containing flat plate and the blank control when the colony of the blank control grows to a preset standard, calculating the hypha growth inhibition rate, establishing a virulence regression equation and calculating an EC50 value; S6, evaluating the activity, namely judging the toxicity level of the bactericide on the red-shell fungus in the strengthening soil according to the EC50 value.
2. The method for evaluating the in-vitro activity of a bactericide for controlling the strong red-shell fungus according to claim 1, wherein in the step S1, the PDA culture medium is prepared from the following raw materials in proportion: Every 1000mL of distilled water contains 5.0g-8.0g of potato starch, 15.0g-25.0g of glucose and 15.0g-25.0g of agar; the sterilization condition is that the temperature is 115-125 ℃ and the time is 20-30 min; The cooling standby temperature is 45-50 ℃.
3. The method for evaluating the in vitro activity of a bactericide for controlling the red-shell fungus of the strong soil according to claim 1, wherein in the step S2, the red-shell fungus of the strong soil is a red-shell fungus LJJZ-1 strain isolated from a stem-based rot disease strain of schisandra chinensis; the conditions of the activation culture are that the temperature is 24-26 ℃ and the dark culture is 5-10 d; the diameter of the standardized bacterial cake is 5mm-10mm.
4. The method for evaluating the in-vitro activity of the bactericide for preventing and treating the red-shell fungus of strong soil according to claim 1, wherein in the step S3, the preparation method of the mother solution of the bactericide to be tested is as follows: For the water-soluble bactericide, directly using sterile water for dissolution and preparation; For the insoluble bactericide, dimethyl sulfoxide is used for dissolution and preparation, and the volume concentration of the dimethyl sulfoxide in the final drug-containing flat plate is controlled to be less than or equal to 0.1%.
5. The method for evaluating the in vitro activity of a bactericide for controlling the strong red-shell fungus according to claim 1, wherein in the step S3, the preset concentration gradient comprises at least one of the following range settings: Aiming at the high-activity bactericide, the concentration gradient range is set to be 0.1mg/L-60.0mg/L; for the moderate-activity bactericide, the concentration gradient range is set to be 1.0mg/L-100.0mg/L; for the low-activity bactericide, the concentration gradient range is set to be 20.0mg/L-1000.0mg/L; 5-7 concentration gradients were set for each sterilant.
6. The method for evaluating the in-vitro activity of the bactericide for preventing and treating the red-shell fungus of the strong soil according to claim 1, wherein in the step S3, the preparation method of the medicine-containing flat plate is that the diluted medicine solution and the PDA culture medium cooled to 45-50 ℃ are mixed uniformly according to the volume ratio of 1 (8-12), and poured into a culture dish for condensation.
7. The method for evaluating the in-vitro activity of a bactericide for controlling the strong red-shell fungus according to claim 1, wherein in the step S4, the inoculation mode is that mycelium faces downwards to be attached to the surface of a culture medium; The temperature of the constant temperature dark condition is 24-26 ℃ and the culture time is 6-8 d.
8. The method for evaluating the in vitro activity of a bactericide against chaetomium robustum according to claim 1, wherein in step S5, the predetermined criterion is that the colony of the blank control grows to be close to the edge of the culture dish; the measuring mode is a crisscross method; the virulence regression equation is established by taking the logarithmic value of the concentration of the medicament as an independent variable and the numerical value of the hypha growth inhibition rate as a dependent variable.
9. The method for evaluating the in-vitro activity of the bactericide for preventing and treating the red-shell fungus of strong soil according to claim 1, wherein in the step S6, specific criteria for evaluating the activity are as follows: when the EC50 value is less than 1.0mg/L, determining that the drug is a high-efficiency drug; determining a moderately active agent when the EC50 value is between 1.0mg/L and 50.0 mg/L; When the EC50 value is greater than 50.0mg/L, it is determined to be an inefficient and ineffective agent.
10. The method for evaluating the in-vitro activity of a bactericide for preventing and treating the red-shell fungus of strong soil according to claim 1, wherein the bactericide to be tested is at least one selected from the group consisting of tebuconazole, pyraclostrobin, kuh-seng osthole, fludioxonil, ethylicin, azoxystrobin, tea saponin, ningnanmycin and amino-oligosaccharin.

Description

In-vitro activity evaluation method of bactericide for preventing and treating red-shell fungus Technical Field The invention relates to the technical field of plant protection, in particular to an in-vitro activity evaluation method of a bactericide for preventing and treating strong soil red shell bacteria. Background Fructus Schisandrae is used as perennial woody vine of Schisandraceae, and is mainly distributed in northeast China. The fruit is rich in lignans, polysaccharide, volatile oil and other active substances, has the pharmacological effects of protecting liver, resisting oxidation, protecting central nervous system and the like, is an important raw material of medicines, health-care products and functional foods, and has higher economic value. However, as the planting scale is enlarged, the occurrence of stem basal rot of schisandra chinensis is increasingly serious, and the stem basal rot becomes a bottleneck for restricting the development of industry. The disease mainly occurs at the junction of the stem base and the rhizome in the surface soil layer, the early stage of the disease appears as a young She Weinian, the cortex of the disease portion is rotted and shed along with the disease development, and the vascular bundle is browned, so that the overground part is dead. Recent researches confirm that the eruption of the stem basal rot of the schisandra chinensis is caused by the strong red-shell fungus, which is the first report that the pathogenic fungus infects the stem basal part of the schisandra chinensis in the global scope, and the pathogenicity is strong and the plant mortality rate is high after the attack. For this newly discovered disease, there is currently no standardized agent screening and evaluation system. The existing screening of the control medicament mainly depends on field efficacy tests, but the mode is greatly influenced by fluctuation of natural environment conditions, and the test period generally spans the whole growing season, so that the screening efficiency is low, and sudden disease epidemic is difficult to rapidly cope with. In production practice, because of lack of drug sensitivity data for the strong red-shelled bacteria, the control work often depends on experience or refers to the medication scheme of other crops, and the blindness not only leads to unstable control effect, but also often causes serious economic loss due to missing of the optimal control window period. In addition, the infection part of the schisandra stem basal rot is hidden, the systemic conductivity and the therapeutic activity of the medicament are difficult to accurately distinguish by simply relying on field experiments, and accurate virulence parameters cannot be provided to balance the efficacy and the safety. The lack of quantitative toxicity data support is easy to cause hidden phytotoxicity caused by over high concentration of the medicament in production, or failure in prevention and control caused by improper selection of the medicament, and has larger ecological risks and economic risks. Therefore, a rapid, accurate and standardized evaluation method for the in-vitro activity of the strong red-shelled turtle shell bactericide is established, and has important significance for scientific prevention and control of schisandra stem basal rot. Disclosure of Invention Aiming at the defects of the prior art, the invention provides an in-vitro activity evaluation method for a bactericide for preventing and treating the red-shell fungus of strong soil, which solves the problems of blindness in medicament selection, long evaluation period and low accuracy caused by lack of a standardized medicament screening system for the red-shell fungus of strong soil in prevention and treatment of stem basal rot of schisandra chinensis. The invention aims at realizing the aim by adopting the following technical scheme that the method for evaluating the in-vitro activity of the bactericide for preventing and treating the strong red-shelled bacteria comprises the following steps: S1, preparing a culture medium, namely preparing a PDA culture medium for the growth of the chaetomium robustum, sterilizing and cooling for later use; s2, preparing a fungus cake, namely inoculating the strong soil red shell fungus to a PDA culture medium for activation culture, and taking a standardized fungus cake with vigorous hypha growth; s3, preparing a medicine-containing flat plate, namely preparing a bactericide to be detected into mother liquor, and respectively adding the mother liquor into the PDA culture medium in the step one according to a preset concentration gradient to prepare medicine-containing flat plates with different concentrations; S4, inoculating and culturing, namely inoculating the standardized bacterial cake prepared in the step S2 to the centers of the drug-containing flat plate and the blank control in the step III, and culturing under the condition of constant temperature and darkness; S5,